Chemically-locked bispecific antibodies

ABSTRACT

Provided are bispecific antibody compounds having the Formula I: 
     
       
         
         
             
             
         
       
     
     wherein, FAB 1 , FAB 2 , and —X— are as defined herein. The provided bispecific antibody compounds can be used a modulators of target molecules, including CD3, PSMA, CD19, CXCR5, CD33, PDL1, VEGFR2, cMet, or Ax1, and are useful in the treatment of one or more conditions.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/257,044, filed Nov. 18, 2015.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 17, 2016, isnamed 126036-00402 SL.txt and is 15,301 bytes in size.

BACKGROUND

Bispecific antibodies are antibodies or antibody-like molecules havingtwo different binding specificities. Because of this unique feature,bispecific antibodies not only connect therapeutics (e.g., T cells anddrugs) with targets (e.g., tumors), but they can also block separatepathogenic mediators. Clinical successes and impressive treatmentprofiles against cancer, autoimmune diseases, and inflammatory diseaseshave been shown. See e.g., MAbs. 2009 November-December; 1(6): 539-547.Given their expanding therapeutic potential, the need for identifyingnew bispecific antibodies remains.

SUMMARY

It has now been found that bispecific antibody compounds having thegeneral Formula I:

or a pharmaceutically acceptable salt thereof, and compositionscomprising these bispecific antibody compounds, wherein, FAB¹, FAB², and—X— are as defined herein, are effective therapeutics (e.g., in thetreatment of cancer. See e.g., FIGS. 9-12.

In addition, the bispecific antibody compounds and compositionsdescribed herein can be manufactured in commercially relevant yields andquantities, utilize digestions on off-the-shelf antibodies or cells(e.g., CHO cells), undergo facile conjugation processes, and elicit theexclusive formation of heterodimers (with a high bispecific antibodyassembly yield). These processes mitigate conventional requirements forextensive protein engineering of each antibody, complex genetictechniques, and laborious biochemical processing

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic illustration of the generation of abispecific antibody compound as described herein.

FIG. 2 illustrates an SDS-PAGE gel analysis of intermediates involved inthe synthesis of an exemplary bispecific antibody.

FIG. 3 illustrates a Hydrophobic Interaction Chromatography (HIC)analysis of bispecific antibody compound 105.

FIG. 4 illustrates simultaneous binding of bispecific antibody compound105 to two antigens on Octet Red.

FIG. 5 illustrates the SDS-PAGE analysis following digestion andpurification from full length IgG1 antibody.

FIG. 6 illustrates a Hydrophobic Interaction Chromatography (HIC)analysis of PSMA F(ab) and CD3 F(ab) following digestion andpurification from full length PSMA IgG1 and full length CD3 IgG1.

FIG. 7 shows a general synthetic scheme for the formation of PSMA/CD3bispecific antibody compounds 106, 107, 108, and 109.

FIG. 8 shows the HIC analysis of aPSMA F(ab) and intermediates aPSMAF(ab)-PEG4-azide and aPSMA F(ab)-PEG8-azide prior to cyclization.

FIG. 9 shows the HIC analysis of αCD3 F(ab) and intermediates αCD3F(ab)-PEG4-DBCO and αCD3 F(ab)-PEG8-DBCO prior to cyclization.

FIG. 10 shows the SDS-PAGE analysis of αPSMA/αCD3 bispecific antibodycompounds 106, 107, 108, and 109.

FIG. 11 shows % killing vs concentration of bispecific antibody compound108 in LnCaP and PC3 Cells.

FIG. 12 shows % killing vs concentration of bispecific antibody compound109 in LnCaP and PC3 Cells.

DETAILED DESCRIPTION

Provided herein are bispecific antibody compounds having the Formula I:

wherein, FAB¹ represents a first Fab fragment; FAB² represents a secondFab fragment; and —X— represents an optionally substituted triazolylcovalently linking FAB¹ and FAB² together.

Definitions

The term “antibody”, as used herein, refers to any immunoglobulin (Ig)molecule comprised of four polypeptide chains, two heavy (H) chains andtwo light (L) chains, or any functional fragment (e.g., a Fab fragment),mutant, variant, or derivation thereof (e.g., a bispecific antibodycompound of Formula I). Such mutant, variant, or derivative antibodyformats are known in the art. In a full-length antibody, each heavychain is comprised of a heavy chain variable region (abbreviated hereinas HCVR or VH) and a heavy chain constant region. The heavy chainconstant region is comprised of three domains, CH1, CH2 and CH3. Eachlight chain is comprised of a light chain variable region (abbreviatedherein as LCVR or VL) and a light chain constant region. The light chainconstant region is comprised of one domain, CL. The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass. In some embodiments, the antibody is a full-lengthantibody. In some embodiments, the antibody is a murine antibody. Insome embodiments, the antibody is a human antibody. In some embodiments,the antibody is a humanized antibody. In other embodiments, the antibodyis a chimeric antibody. Chimeric and humanized antibodies may beprepared by methods well known to those of skill in the art includingCDR grafting approaches (see, e.g., U.S. Pat. Nos. 5,843,708; 6,180,370;5,693,762; 5,585,089; and 5,530,101), chain shuffling strategies (see,e.g., U.S. Pat. No. 5,565,332; Rader et al. (1998) PROC. NAT'L. ACAD.SCI. USA 95: 8910-8915), molecular modeling strategies (U.S. Pat. No.5,639,641)). In one embodiment, the antibodies described herein (e.g.,FAB¹ and FAB²) do not comprise a hinge region.

The term “bispecific antibody” refers to any immunoglobulin (Ig)molecule comprised of four polypeptide chains, two heavy (H) chains andtwo light (L) chains, or any functional fragment (e.g., a Fab fragment),mutant, variant, or derivation thereof (e.g., a bispecific antibodycompound of Formula I), which can bind to two different epitopes. In oneembodiment, the bispecific antibody binds to two different epitopes onthe same antigen. In one embodiment, the bispecific antibody binds toepitopes on two different antigens. In one embodiment, the bispecificantibody described herein is of the Formula I, wherein FAB¹ and FAB² donot comprise a hinge region.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Such antibodyembodiments may also be bispecific, dual specific, or multi-specificformats; specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) an Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) and a dAbfragment (Ward et al. (1989) NATURE 341: 544-546; and Winter et al., PCTPublication No. WO 90/05144 A1).

Various techniques are known in the art for the production of antibodyfragments. Antibody fragments may be derived via proteolytic digestionof intact antibodies (see, e.g., Morimoto et al., J Biochem Biophys.Method. 24:107-117 (1992); and Brennan et al., Science 229:81 (1985)).Antibody fragments may also be produced directly by recombinant hostcells. For example, Fab, Fv and scFv antibody fragments can all beexpressed in and secreted from E. coli, thus allowing the facileproduction of large amounts of these fragments. Antibody fragments canbe isolated from the antibody phage libraries discussed below. Suchfragments may be conjugated as described herein.

The terms “Fab” or “FAB” or “Fab fragment”, as used interchangeablyherein, refers to an antibody fragment which is a monovalent fragmenthaving VL, VH, CL and CH1 domains. Unless otherwise specified, a Fabdoes not contain an Fc region or a hinge region linking the CH1 and CH2domains of the heavy chain. A F(ab)2 refers to the bispecific antibodycompound of Formula I. In one aspect, the terms “FAB¹” or “FAB²” referto a Fab fragment of an antibody. In one aspect, FAB¹ and FAB² do notcomprise a hinge region.

The term “human antibody”, as used herein, refers to a recombinantantibody having one or more variable and constant regions derived fromhuman immunoglobulin sequences. In one embodiment, all of the variableand constant domains are derived from human immunoglobulin sequences (afully human antibody). A human antibody may be prepared in a variety ofways, examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

The term “humanized antibody” refers to an antibody comprising at leastone chain comprising variable region framework residues substantiallyfrom a human antibody chain (referred to as the acceptor immunoglobulinor antibody) and at least one CDR substantially from a non-human, e.g.,a mouse, antibody, (referred to as the donor immunoglobulin orantibody). See, methods of making described in Queen et al., Proc. Natl.Acad. Sci. USA 86:10029 10033 (1989), U.S. Pat. Nos. 5,530,101,5,585,089, 5,693,761, WO 90/07861, and U.S. Pat. No. 5,225,539, each ofwhich is incorporated by reference herein. The constant region(s), ifpresent, can also be substantially or entirely from a humanimmunoglobulin. Methods of making humanized antibodies are known in theart. See, e.g., U.S. Pat. No. 7,256,273, incorporated by referenceherein. In one embodiment, certain amino acids in the framework andconstant domains of the heavy and/or light chains of a non-human speciesantibody are mutated to produce the humanized antibody. Further examplesof how to make humanized antibodies may be found in U.S. Pat. Nos.6,054,297; 5,886,152; and 5,877,293, each of which is incorporated byreference herein.

An “epitope”, as used herein, is the portion of a molecule that is boundby an antibody. In one embodiment, an epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The term “isolated” refers to a molecule that is identified andseparated from at least one contaminant molecule with which it isordinarily associated in the natural source of the molecule. Preferably,the isolated molecule is free of association with all components withwhich it is naturally associated. In one aspect, the antibodiesdescribed are isolated.

As used herein, “substituted triazolyl” refers to a triazoyl group thatis substituted with one or more groups that do not substantially alterconditions which allow for the production, detection, and, in certainembodiments, the recovery, purification, and use for one or more of thebispecific antibody compounds disclosed herein The point of attachmentcan be on any substitutable position and, include, e.g., 1,2,3-triazolyl(e.g., substituted 1,4; 1,5; 4,5; and 1,4,5) and 1,2,4-triazolyl (e.g.,substituted 3,4; 3,5; 4,5; and 3,4,5).

Oxo refers to the functional group “═O” (a substituent oxygen atomconnected to another atom by a double bond).

The term “alkyl” means saturated straight-chain or branched monovalenthydrocarbon radical. As used herein a “(C₂-C₂₀)alkyl” group is means aradical having from 2 to 20 carbon atoms in a linear or branchedarrangement. Where defined, alkyl groups may be interrupted by one ormore heteroatoms selected from O, N, and S.

The term “alkyne” refers to an unsaturated hydrocarbon containing atleast one carbon-carbon triple bond between two carbon atoms. Terminalalkyne means that the carbon-carbon triple bond between two carbon atomsis at the end of the carbon chain e.g., as in where there is at leastone hydrogen atom bonded to a triply bonded carbon atom (e.g.,pent-1-yne).

The term “aryl” refers to an aromatic monocyclic or bicyclic carbon ringsystem having, unless otherwise specified, a total of 6 to 14 ringmembers. The term “aryl” may be used interchangeably with the term “arylring”, “aryl group”, “aryl moiety,” or “aryl radical”. Also includedwithin the scope of the term “aryl”, as it is used herein, is a group inwhich an aromatic carbon ring is fused to one or more carbocyclyl rings,e.g., tetrahydronaphthalenyl. In certain embodiments of the presentdisclosure, “aryl” refers to an aromatic ring system which includes, butis not limited to, phenyl (abbreviated as “Ph”), naphthyl and the like.It will be understood that when specified, optional substituents on anaryl group (e.g., in the case of an optionally substituted aryl or arylwhich is optionally substituted) may be present on any substitutableposition, i.e., any ring carbon substituted with hydrogen.

The term “heteroaryl” used alone or as part of a larger moiety as in“heteroarylalkyl”, “heteroarylalkoxy”, or “heteroarylaminoalkyl”, refersto a 5-10-membered aromatic radical containing 1-4 heteroatoms selectedfrom N, quaternary ammonium cation, 0, and S, and includes, for example,thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl,tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring”, “heteroarylgroup”, or “heteroaromatic”. Nonlimiting examples include indolyl,indazolyl, benzimidazolyl, benzthiazolyl, pyrrolopyridinyl, quinolyl,quinazolinyl, and quinoxalinyl. It will be understood that whenspecified, optional substituents on a heteroaryl group may be present onany substitutable position (carbon and nitrogen).

The term “carbocyclyl” as used herein, means a monocyclic, bicyclic(e.g., a bridged or spiro bicyclic ring), polycyclic (e.g., tricyclic ormore), or fused hydrocarbon ring system that is completely saturated orthat contains one or more units of partial unsaturation, but where thereis no aromatic ring. Cycloalkyl is a completely saturated carbocycle.Monocyclic carbocyclyl groups include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, cycloheptenyl, and cyclooctyl. Bridged bicyclic carbocyclylgroups include, without limitation, bicyclo[3.2.1]octane,bicyclo[2.2.1]heptane, bicyclo[3.1.0]hexane, and the like. Spirobicyclic carbocyclyl groups include, e.g., spiro[3.6]decane,spiro[4.5]decane, and the like. Fused carbocyclyl rings include, e.g.,decahydronaphthalene, octahydropentalene, and the like. Polycycliccarbocyclyl rings include e.g., bicyclo[6.1.0]nonane and1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazole.It will be understood that when specified, optional substituents on acarbocyclyl (e.g., in the case of an optionally substituted carbocyclylor carbocyclyl which is substituted) may be present on any substitutableposition and, include, e.g., the position at which the carbocyclyl groupis attached.

The term “heterocyclyl” means a 3-12 membered (e.g., a 4-, 5-, 6-7- and8-membered) saturated or partially unsaturated heterocyclic ringcontaining 1 to 4 heteroatoms independently selected from N, O, and S.It can be mononcyclic, bicyclic (e.g., a bridged, fused, or Spirobicyclic ring), or polycyclic (e.g., tricyclic or more). The terms“heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclicgroup”, “heterocyclic moiety”, and “heterocyclic radical”, are usedinterchangeably herein. A heterocyclyl ring can be attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure. Examples of such saturated or partially unsaturatedheterocyclic radicals include, without limitation, tetrahydrofuranyl,tetrahydrothienyl, terahydropyranyl, pyrrolidinyl, pyridinonyl,pyrrolidonyl, piperidinyl, oxazolidinyl, piperazinyl, dioxanyl,dioxolanyl, morpholinyl, dihydrofuranyl, dihydropyranyl,dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl,3-azabicyclo[3.1.0]hexanyl, 2-oxa-6-azaspiro[3.3]heptanyl,1-azaspiro[4.5]decane, and tetrahydropyrimidinyl. The term“heterocyclyl” also includes, e.g., unsaturated heterocyclic radicalsfused to another unsaturated heterocyclic radical or aryl or heteroarylring, such as for example, tetrahydronaphthyridine, indolinone,dihydropyrrolotriazole, imidazopyrimidine, quinolinone, anddioxaspirodecane. Examples of polycyclic (e.g., tricyclic or more)heterocyclyl include, without limitation,5,6,11,12-tetrahydrodibenzo[b,f]azocine and8,9-dihydro-1H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocine. It will alsobe understood that when specified, optional substituents on aheterocyclyl group may be present on any substitutable position and,include, e.g., the position at which the heterocyclyl is attached (e.g.,in the case of an optionally substituted heterocyclyl or heterocyclylwhich is optionally substituted).

The term “spiro” refers to two rings that share one ring atom (e.g.,carbon).

The term “fused” refers to two rings that share two adjacent ring atoms.

The term “bridged” refers to two rings that share at least three ringatoms.

As described herein, the moieties present on the substituted triazolylmay be further substituted or contain “optionally substituted” moieties.For example, optionally substituted alkyl, optionally substitutedpyrazolyl, an optionally substituted carbocyclic, an optionallysubstituted multi-cyclic heterocyclic ring system, etc. Unless otherwiseindicated, an “optionally substituted” group may have a suitablesubstituent that results in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers to groupsthat are not substantially altered when subjected to conditions to allowfor their production, detection, and, in certain embodiments, theirrecovery, purification, and use for one or more of the purposesdisclosed herein.

In one aspect, suitable substituents for an optionally substituted orsubstituted alkyl, carbocyclyl, or heterocyclyl group are those which donot substantially diminish the yield of the bispecific antibodycompound. Examples include halogen, CN, OR^(c), —NR^(d)R^(e),—S(O)_(i)R^(c), —NR^(c)S(O)₂R^(c), —S(O)₂NR^(d)R^(e), —C(═O)OR^(c),—OC(═O)OR^(c), —OC(═O)R^(c), —OC(═S)OR^(c), —C(═S)OR^(c), —O(C═S)R^(c),—C(═O)NR^(d)R^(e), —NR^(c)C(═O)R^(c), —C(═S)NR^(d)R^(e),—NR^(c)C(═S)R^(c), —NR^(c)(C═O)OR^(c), —O(C═O)NR^(d)R^(e),—NR^(c)(C═S)OR^(c), —O(C═S)NR^(d)R^(e), —NR^(c)(C═O)NR^(d)R^(e),—NR^(c)(C═S)NR^(d)R^(e), —C(═S)R^(c), —C(═O)R^(c), (C₁-C₆)alkyl,cycloalkyl, —(CH₂)₁₋₄-cycloalkyl, heterocyclyl, —(CH₂)₁₋₄-heterocyclyl,aryl, —NHC(═O)-heterocyclyl, —NHC(═O)-cycloalkyl, —(CH₂)₁₋₄-aryl,heteroaryl or —(CH₂)₁₋₄-heteroaryl, wherein each of said (C₁-C₆)alkyl,cycloalkyl, —(CH₂)₁₋₄-cycloalkyl, heterocyclyl, —(CH₂)₁₋₄-heterocyclyl,aryl, —(CH₂)₁₋₄-aryl, heteroaryl and —(CH₂)₁₋₄-heteroaryl are optionallysubstituted with halogen, OR^(c), —NO₂, —CN, —NR^(c)C(═O)R^(c),—NR^(d)R^(e), —S(O)_(k)R^(c), —C(═O)OR^(c), —C(═O)NR^(d)R^(e),—C(═O)R^(c), (C₁-C₃)alkyl, halo(C₁-C₃)alkyl, (C₁-C₃)alkoxy(C₁-C₃)alkyl,(C₁-C₃)alkoxy, and halo(C₁-C₃)alkoxy, wherein R^(c) is hydrogen or(C₁-C₆)alkyl optionally substituted with 1 to 3 halogen; R^(d) and R^(e)are each independently selected from hydrogen and (C₁-C₆)alkyl; and k is0, 1 or 2. Suitable substituents for optionally substituted alkyl,carbocyclyl, and heterocyclyl also include oxo (═O).

The bispecific antibody compounds described herein may be present in theform of pharmaceutically acceptable salts. For use in medicines,pharmaceutically acceptable salts refer to non-toxic pharmaceuticallyacceptable salts. Pharmaceutically acceptable salt forms includepharmaceutically acceptable acidic/anionic or basic/cationic salts.Suitable pharmaceutically acceptable acid addition salts of thecompounds described herein include e.g., salts of inorganic acids (suchas hydrochloric acid, hydrobromic, phosphoric, nitric, and sulfuricacids) and of organic acids (such as, acetic acid, benzenesulfonic,benzoic, methanesulfonic, and p-toluenesulfonic acids). Suitablepharmaceutically acceptable basic salts include e.g., ammonium salts,alkali metal salts (such as sodium and potassium salts) and alkalineearth metal salts (such as magnesium and calcium salts).

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed, i.e., therapeutic treatment.In other embodiments, treatment may be administered in the absence ofsymptoms. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors),i.e., prophylactic treatment. Treatment may also be continued aftersymptoms have resolved, for example to prevent or delay theirrecurrence.

The terms “subject” and “patient” may be used interchangeably, and meansa mammal in need of treatment, e.g., companion animals (e.g., dogs,cats, and the like), farm animals (e.g., cows, pigs, horses, sheep,goats and the like) and laboratory animals (e.g., rats, mice, guineapigs and the like). Typically, the subject is a human in need oftreatment.

Bispecific Antibody Compounds and Methods of Making Same

In a first exemplary embodiment, the bispecific antibody compounds ofFormula I are of the Formula II, IIa, III, or Ma:

wherein R¹ and R² are each independently a substituted alkyl; ring A isa substituted carbocyclyl or substituted heterocyclyl; and R^(a) andR^(b) are each independently selected from

wherein Q, T, and V are each independently N or CH; “

” indicates the points of attachment to FAB¹ or FAB² and “ ” indicatesthe point of attachment to R¹ or R², and wherein the remaining variablesand values are as described for Formula I.

In a second exemplary embodiment, Ring A in Formula I, III, or Ma is asubstituted bicyclic or polycyclic carbocyclyl or a substitutedpolycyclic heterocyclyl, wherein the remaining variables and values areas described for Formula I or the first exemplary embodiment.

In a third exemplary embodiment, Ring A is

wherein the dashed bonds indicate the points of attachment to thetriazolyl and the wavy bond indicates the attachment to R², and whereinthe remaining variables and values are as described for Formula I or thefirst or second exemplary embodiment.

In a fourth exemplary embodiment, R¹ and R² in Formula I, II, IIa, III,or IIIc are each independently an optionally substituted (C₂-C₃₀)alkyloptionally interrupted with one or more heteroatoms selected from N, O,and S, wherein the remaining variables and values are as described forFormula I or the first, second, or third exemplary embodiment.

In a fifth exemplary embodiment, R¹ and R² in Formula I, II, IIa, III,or IIIc are each independently a substituted (C₂-C₃₀)alkyl optionallyinterrupted with one or more heteroatoms selected from N and O, whereinthe remaining variables and values are as described for Formula I or thefirst, second, third, or fourth exemplary embodiment.

In a sixth exemplary embodiment, R¹ and R² in Formula I, II, IIa, III,or IIIc are each independently a (C₂-C₃₀)alkyl interrupted with at leastone O and at least one N, and substituted with at least one oxo, whereinthe remaining variables and values are as described for Formula I or thefirst, second, third, fourth, or fifth exemplary embodiment.

In a seventh exemplary embodiment, R¹ and R² in Formula I, II, IIa, III,or Ma are each independently selected from

the wavy lines indicate the points of attachment to R^(a) or R^(b); thedashed lines indicated the points of attachment to the triazolyl or ringA; and p and w independently are integers from 1 to 8, wherein theremaining variables and values are as described for Formula I or thefirst, second, third, fourth, fifth, or sixth exemplary embodiment.

In an eighth exemplary embodiment, R¹ in Formula I, II, IIa, III, or Mais selected from

wherein the wavy lines indicate the points of attachment to R^(a); andthe dashed lines indicated the points of attachment to the triazolyl,and wherein the remaining variables and values are as described forFormula I or the first, second, third, fourth, fifth, sixth, or seventhexemplary embodiment.

In a ninth exemplary embodiment, R² in Formula I, II, IIa, III, or Ma isselected from

the wavy lines indicate the points of attachment to R^(a); and thedashed lines indicated the points of attachment to the triazolyl or ringA, wherein the remaining variables and values are as described forFormula I or the first, second, third, fourth, fifth, sixth, seventh, oreighth exemplary embodiment.

In a tenth exemplary embodiment, R^(a) and R^(b) in Formula I, II, IIa,III, or IIIc are bound to FAB¹ and FAB² through native cysteines of FAB¹and FAB², wherein the remaining variables and values are as describedfor Formula I or the first, second, third, fourth, fifth, sixth,seventh, eighth, or ninth exemplary embodiment. Alternatively, R^(a) andR^(b) in Formula I, II, IIa, III, or Ma are bound to FAB¹ and FAB²through native cysteines that are responsible for forming interchaindisulfide bonds of FAB¹ and FAB², wherein the remaining variables andvalues are as described for Formula I or the first, second, third,fourth, fifth, sixth, seventh, eighth, or ninth exemplary embodiment.

In an eleventh exemplary embodiment, the bispecific antibody compound ofFormula I is of the formula:

or a pharmaceutically acceptable salt thereof, wherein FAB¹ and FAB² areconnected to the pyrrolidine-dione through native cysteine residues.

In a thirteenth embodiment, the bispecific antibody compound of FormulaI is of the formula:

or a pharmaceutically acceptable salt thereof, wherein FAB¹ and FAB² areconnected to the pyrrolidine-dione through native cysteine residues.

In a fourteenth embodiment, FAB¹ and FAB² in any one of the bispecificantibody compounds described herein are each independently selected froma Fab fragment comprising a CD3 binding region and a Fab fragmentcomprising a PSMA binding region.

The bispecific antibody compounds described herein can be readilyprepared according to the following reaction schemes and examples, ormodifications thereof, using readily available starting materials,reagents and conventional synthesis procedures. In addition, one canrefer to the following references for suitable methods of synthesis asdescribed in March, Advanced Organic Chemistry, 3rd edition, John Wiley& Sons, 1985, Greene and Wuts, Protective Groups in Organic Synthesis,2^(nd) edition, John Wiley & Sons, 1991, and Richard Larock,Comprehensive Organic Transformations, 4^(th) edition, VCH publishersInc., 1989

Bispecific antibody compounds of Formula I may be prepared according tothe general reaction scheme shown in FIG. 1. In a first process, the Fcfragment along with hinge region of full length FAB is removed viadigestion, as described, for example, in FIG. 1 (such as papaindigestion). FAB¹ and FAB² are then selectively reduced to form Fabfragments. Functional moieties, X or Y (where one X or Y is an azide(N₃) and the other X or Y is an alkyne) are introduced into each Fab viaa cysteine-based conjugation, leading to chemically modified Fabfragments, respectively in FIG. 1. The functional moieties X and Y arepreferably introduced via conjugation to cysteine residues within theconstant region of each Fab fragment, i.e., the light chain CL regionand heavy chain CH1 constant region.

In order to achieve the chemical linkage, cysteine residues within theCH1 of the heavy chain and the CL of the light chain are reduced. In oneembodiment, native cysteines that form the interchain disulfide bondsare reduced and used to chemically modify the Fab fragment as describedherein. In one embodiment, the starting antibodies may containmodifications within the heavy and light chain constant regions (CH1 andCL, respectively) where additional cysteine residues are introduced.

Two Fab fragments are then linked together through a chemical ligationbetween X and Y moieties, to form X-Y, which correlates to variable“—X—” in the bispecific antibody compounds of Formula I.

For example, in instances where the bispecific antibody compounds arerepresented by Formula II, the azide could be attached to R¹ and theterminal alkyne could be attached to R², where ligation would occur toform the triazolyl. See Scheme 1 below.

It will be understood that the reverse could also be employed where theazide is attached to R² and the terminal alkyne is on R¹ to formbispecific antibody compounds represented by Formula IIa.

In another example, in instances where the bispecific antibody compoundsare represented by Formula III, the azide could be attached to R¹ andthe alkyne could be attached to R², where ligation would occur to formthe triazolyl. See Scheme 2 below.

It will be understood that the reverse could also be employed where theazide is attached to R² and the alkyne is on R¹ to form bispecificantibody compounds represented by Formula Ma.

In one aspect, the FAB¹ and FAB² are each capable of binding twodifferent epitopes on the same or on different antigens. In oneembodiment, FAB¹ and FAB² bind to two different epitopes on the sameantigen. In one embodiment, FAB¹ and FAB² bind to two differentantigens.

In one embodiment, FAB¹ and FAB² are each independently IgG1 or IgG4isotypes. In one embodiment, FAB¹ and FAB² are each IgG1 isotypes. Inone embodiment, FAB¹ and FAB² are each IgG4 isotypes. In one embodiment,FAB¹ is an IgG1 isotype and FAB² is an IgG4 isotype. In anotherembodiment, FAB² is an IgG1 isotype and FAB¹ is an IgG4 isotype. In oneembodiment, the bispecific antibody compounds described herein bind to atarget molecule selected from the group consisting of CD3, PSMA, CD19,CXCR5, CD33, PDL1, VEGFR2, cMet, and Ax1. In one embodiment, thebispecific antibody compounds described herein bind to a pair ofantigens selected from the following group: CD3-PSMA, CD3-CD19,CD3-CXCR5, CD3-CD33, PDL1-VEGFR2, PDL1-cMet, PDL1-Ax1.

In one embodiment, the bispecific antibody compounds described hereinbind to two epitopes on CD3 or binds to CD3 and another target molecule.In one embodiment, the bispecific antibody compounds described hereincomprise a CD3 binding region corresponding to the CD3 binding portion,e.g., a Fab fragment, of BLINCYTO (Blinatumomab; Amgen). In anotherembodiment, the bispecific antibody compounds described herein comprisea Fab fragment corresponding to anti-CD3 antibodies HuM291, UCHT1, orOKT3). In another embodiment, the bispecific antibody compoundsdescribed herein comprise a Fab fragment corresponding to anti-CD3antibodies comprising a heavy chain region comprising the amino acidsequence QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 5);or an heavy chain comprising SEQ ID NO: 5 and light chain regioncomprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6); or a light chain regioncomprising SEQ ID NO: 6.

In another embodiment, the bispecific antibody compounds describedherein comprise a Fab fragment corresponding to anti-CD3 antibodiescomprising a heavy chain variable region (HCVR) comprising the aminoacid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGT LVTVSS (SEQ IDNO: 9); or an heavy chain comprising SEQ ID NO: 9 and light chain regioncomprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6); or a light chain regioncomprising SEQ ID NO: 6. In another embodiment, the bispecific antibodycompounds described herein comprise a Fab fragment corresponding toanti-CD3 antibodies comprising a heavy chain region comprising the aminoacid sequence EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMetNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMetNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO:7); or a heavy chain region comprising SEQ ID NO: 7 and light chainregion comprising the amino acid sequence of

DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8). In anotherembodiment, the bispecific antibody compounds described herein comprisea Fab fragment corresponding to anti-CD3 antibodies comprising a heavychain variable region comprising the amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMetNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMetNSLRAEDTAVYYCARSGYYGDSDWYFDVW GQGTLVTVSS(SEQ ID NO: 10); or an HCVR comprising SEQ ID NO: 10 and light chainregion comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQURNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8).

In one embodiment, the bispecific antibody compounds described hereinbind to two epitopes on prostate specific membrane antigen protein(PSMA) or binds to PSMA and another target molecule. In one embodiment,the bispecific antibody compounds described herein comprise a Fabfragment corresponding to antibodies 3D8, 4D4, and/or 3E11, which aredescribed in US 2007/0031438, the contents of which are incorporated byreference herein.

In one embodiment, the bispecific antibody compounds described hereinbind to two epitopes on CD19 or binds to CD19 and another targetmolecule. In one embodiment, the bispecific antibody compounds describedherein comprise a CD19 binding region corresponding to the CD3 bindingportion, e.g., a Fab fragment, of BLINCYTO (Blinatumomab; Amgen).

In one embodiment, the bispecific antibody compounds described hereinbind to two epitopes on CXCR5 or binds to CXCR5 and another targetmolecule. In one embodiment, the bispecific antibody compounds describedherein comprises a Fab fragment(s) corresponding to anti-CXCR5antibodies which are described in U.S. patent application Ser. No.14/825,144 filed on Aug. 12, 2015, the contents of which areincorporated by reference herein.

In one embodiment, the bispecific antibody compounds described hereinbind to two epitopes on CD33 or binds to CD33 and another targetmolecule.

In one embodiment, the bispecific antibody compounds described hereinbind to two epitopes on PDL1 or binds to PDL1 and another targetmolecule. In one embodiment, the bispecific antibody compounds describedherein comprise a Fab fragment corresponding to anti-PDL-1 antibodieswhich are described in US 2013/0323249 and WO 2013/181634, the contentsof which are each incorporated by reference herein. In one embodiment,the bispecific antibody compounds described herein comprise amino acidsequences corresponding to the Fab fragment of anti-PDL-1 antibodyH6B1L, as described in US 2013/0323249, the contents of which areincorporated by reference herein.

In one embodiment, the bispecific antibody compounds described hereincomprise a Fab fragment corresponding to anti-PDL-1 antibodiescomprising a heavy chain variable region comprising the amino acidsequence of QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLV TVSS (SEQ IDNO: 1), or a HCVR comprising the CDR sequences described in SEQ ID NO:1, and a light chain variable region comprising the amino acid sequenceof SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 2), or aLCVR comprising the CDR sequences described in SEQ ID NO: 2.

In one embodiment, the bispecific antibody compounds described hereinbinds to two epitopes on VEGFR2 or binds to VEGFR2 and another targetmolecule. In one embodiment, the bispecific antibody compounds describedherein comprises a Fab fragment corresponding to anti-VEGFR2 antibodieswhich are described in US 2014/0294827 and WO 2013/149249, the contentsof which are each incorporated by reference herein. In one embodiment,the bispecific antibody compounds described herein comprise amino acidsequences corresponding to the Fab fragment of anti-VEGFR2 antibodyVK-B8, as described in US 2014/0294827, the contents of which areincorporated by reference herein.

In one embodiment, the bispecific antibody compounds described hereincomprise a Fab fragment corresponding to anti-VEGFR2 antibodiescomprising a heavy chain variable region comprising the amino acidsequence of MAQVQLVQSGAEVKKPGSSVKVSCKAYGGTFGSYGVSWVRRAPGQGLEWMGRLIPIFGTRDYAQKFQGRVTLTADESTNTAYMELSSLRSEDTAVYYCARDGDYYGSGSYYGMD VWGQGTLVTVSS(SEQ ID NO: 3), or a HCVR comprising the CDR sequences described in SEQID NO: 3, and a light chain variable region comprising the amino acidsequence of ETTLTQSPATLSVSPGERATVSCRASQSLGSNLGWFQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNDWPITFGQGTRLEIK (SEQ ID NO: 4), or aLCVR comprising the CDR sequences described in SEQ ID NO: 4.

In one embodiment, the bispecific antibody compounds described hereinbind to two epitopes on cMet or binds to cMet and another targetmolecule. In one embodiment, the bispecific antibody compounds describedherein comprise a Fab fragment corresponding to anti-cMet antibodieswhich are described in U.S. patent application Ser. No. 13/924,492 andPCT WO 2013/192594, the contents of which are incorporated by referenceherein.

In one embodiment, the bispecific antibody compounds described hereinbinds to two epitopes on Ax1 or binds to Ax1 and another targetmolecule.

Fab fragments used in the bispecific antibody compounds described hereinmay be made using standard recombinant methods known in the art. In oneembodiment, full length antibodies (i.e., an antibody comprising a Fabregion, a hinge region and an Fc region) are produced and subsequentlydigested to provide Fab fragments for use in the bispecific antibodycompounds described herein. Alternatively, Fab fragments are produced inhost cells, which eliminates the need to digest a full length antibody.

Production methods described herein are applicable to full lengthantibodies and fragments thereof, including Fab fragments.

Recombinant antibody production is known in the art. For example, forrecombinant production of an antibody, the nucleic acid encoding it isisolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The choice of vector depends inpart on the host cell to be used. Generally, preferred host cells are ofeither prokaryotic or eukaryotic (generally mammalian) origin.

In one embodiment, an antibody is produced using prokaryotic cells.Sequences encoding the polypeptides are inserted into a recombinantvector capable of replicating and expressing heterologouspolynucleotides in prokaryotic hosts. Many vectors that are availableand known in the art can be used. Selection of an appropriate vectorwill depend mainly on the size of the nucleic acids to be inserted intothe vector and the particular host cell to be transformed with thevector. Each vector contains various components, depending on itsfunction (amplification or expression of heterologous polynucleotide, orboth) and its compatibility with the particular host cell in which itresides. The vector components generally include, but are not limitedto: an origin of replication, a selection marker gene, a promoter, aribosome binding site (RBS), a signal sequence, the heterologous nucleicacid insert and a transcription termination sequence.

Prokaryotic host cells suitable for expressing antibodies includeArchaebacteria and Eubacteria, such as Gram-negative or Gram-positiveorganisms. Examples of useful bacteria include Escherichia (e.g., E.coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species(e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans,Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. Inone embodiment, gram-negative cells are used. In one embodiment, E. colicells are used as hosts. Examples of E. coli strains include strainW3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington,D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCCDeposit No. 27,325) and derivatives thereof, including strain 33D3having genotype W3110 fhuA (tonA) ptr3 lac Iq lacL8 ompT (nmpc-fepE)degP41 kan.sup.R (U.S. Pat. No. 5,639,635). Other strains andderivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E.coli 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are alsosuitable. These examples are illustrative rather than limiting. Methodsfor constructing derivatives of any of the above-mentioned bacteriahaving defined genotypes are known in the art and described in, forexample, Bass et al., Proteins, 8:309-314 (1990). It is generallynecessary to select the appropriate bacteria taking into considerationreplicability of the replicon in the cells of a bacterium. For example,E. coli, Serratia, or Salmonella species can be suitably used as thehost when well-known plasmids such as pBR322, pBR325, pACYC177, orpKN410 are used to supply the replicon.

Prokaryotic host cells are transformed with the above-describedexpression vectors and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. Transformationmeans introducing DNA into the prokaryotic host so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride is generally used for bacterial cells thatcontain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the bispecific antibody compoundsdescribed herein are grown in media known in the art and suitable forculture of the selected host cells. Examples of suitable media includeluria broth (LB) plus necessary nutrient supplements. In someembodiments, the media also contains a selection agent, chosen based onthe construction of the expression vector, to selectively permit growthof prokaryotic cells containing the expression vector. For example,ampicillin is added to media for growth of cells expressing ampicillinresistant gene.

The expressed antibody proteins described herein are secreted into andrecovered from the periplasm of the host cells. Protein recoverytypically involves disrupting the microorganism, generally by such meansas osmotic shock, sonication or lysis. Once cells are disrupted, celldebris or whole cells may be removed by centrifugation or filtration.The proteins may be further purified, for example, by affinity resinchromatography. Alternatively, proteins can be transported into theculture media and isolated therein. Cells may be removed from theculture and the culture supernatant being filtered and concentrated forfurther purification of the proteins produced. The expressedpolypeptides can be further isolated and identified using commonly knownmethods such as polyacrylamide gel electrophoresis (PAGE) and Westernblot assay.

Alternatively, antibody production is conducted in large quantity by afermentation process. Various large-scale fed-batch fermentationprocedures are available for production of recombinant proteins.Large-scale fermentations have at least 1000 liters of capacity,preferably about 1,000 to 100,000 liters of capacity. These fermentorsuse agitator impellers to distribute oxygen and nutrients, especiallyglucose (the preferred carbon/energy source). Small scale fermentationrefers generally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

Antibodies may also be produced in eukaryotic host cells. For eukaryoticexpression, the vector components are known in the art and generallyinclude, but are not limited to, one or more of the following, a signalsequence, an origin of replication, one or more marker genes, andenhancer element, a promoter, and a transcription termination sequence.

Eukaryotic host cells are transformed with expression or cloning vectorsfor antibody production and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences. Suitable hostcells for cloning or expressing the DNA in the vectors (i.e., DNAencoding an antibody) include higher eukaryote cells described herein,including vertebrate host cells. Propagation of vertebrate cells inculture (tissue culture) has become a routine procedure. Examples ofuseful mammalian host cell lines are monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

The host cells used to produce the antibodies described herein may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or U.S. Pat. No. 5,122,469; WO 90/03430; WO 87/00195; or U.S.Pat. No. Re. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

One produced, the antibody produced herein is further purified to obtainpreparations that are substantially homogeneous for further assays anduses. Standard protein purification methods known in the art can beemployed. The following procedures are exemplary of suitablepurification procedures: fractionation on immunoaffinity or ion-exchangecolumns, ethanol precipitation, reverse phase HPLC, chromatography onsilica or on a cation-exchange resin such as DEAE, chromatofocusing,SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, forexample, Sephadex G-75.

In one embodiment, Protein A may be used to purify a full lengthantibody prior to digestion to obtain Fab fragments used in thebispecific antibody compounds of Formula I. The suitability of Protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human immunoglobulinscontaining 1, 2, or 4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman 3 (Guss et al., EMBO J. 5:15671575 (1986)).

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized is preferably a column comprising a glass or silicasurface, more preferably a controlled pore glass column or a silicicacid column. In some applications, the column has been coated with areagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants. The solid phase is then washed to removecontaminants non-specifically bound to the solid phase. Finally theantibody of interest is recovered from the solid phase by elution.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

Antibodies may be identified using any number of techniques known in theart.

Preferably, the antibody is a monoclonal antibody. Monoclonal antibodiesare obtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translational modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. For example, the monoclonal antibodiesmay be made using the hybridoma method first described by Kohler et al.,Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567).

Monoclonal antibodies may also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567, and as described above.DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, inorder to synthesize monoclonal antibodies in such recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, antibodies can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991)and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe theisolation of murine and human antibodies, respectively, using phagelibraries. Subsequent publications describe the production of highaffinity (nM range) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10:779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

In certain embodiment, the antibodies described herein may be humanizedor human antibodies. Humanized forms of non-human (e.g., murine)antibodies are chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementarity determining region (CDR) (HVR as used herein) ofthe recipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomain, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann etal., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

Recombinant human antibodies can be generated using methods known in theart. For example, transgenic animals (e.g., mice) may be produced thatare capable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);U.S. Pat. No. 5,591,669 and WO 97/17852.

Alternatively, phage display technology can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors. McCafferty et al.,Nature 348:552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227: 381(1991). According to this technique, antibody V domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B-cell.Phage display can be performed in a variety of formats, reviewed in,e.g., Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol.3:564-571 (1993). Several sources of V-gene segments can be used forphage display. Clackson et al., Nature 352:624-628 (1991) isolated adiverse array of anti-oxazolone antibodies from a small randomcombinatorial library of V genes derived from the spleens of immunizedmice. A repertoire of V genes from unimmunized human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffithet al., EMBO J. 12:725-734 (1993). See also, U.S. Pat. Nos. 5,565,332and 5,573,905.

The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human monoclonal antibodies (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) andBoerner et al., J. Immunol. 147(1): 86-95 (1991). Similarly, humanantibodies can be made by introducing human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016 and in the following scientific publications: Marks et al.,Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859(1994); Morrison, Nature 368: 812-13 (1994), Fishwild et al., NatureBiotechnology 14: 845-51 (1996), Neuberger, Nature Biotechnology 14: 826(1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).Finally, human antibodies may also be generated in vitro by activated Bcells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Specific examples of the bispecific antibody compounds are provided inthe EXEMPLIFICATION. Pharmaceutically acceptable salts as well as theneutral forms of these bispecific antibody compounds are includedherein.

In certain embodiments, the present disclosure provides a method oftreating a patient (e.g., a human) with a disorder mediated by atherapeutic target, e.g., CD3, PSMA, CD19, CXCR5, CD33, PDL1, VEGFR2,cMet, and Ax1, comprising the step of administering to the patient aneffective amount of the bispecific antibody compound as describedherein, or a composition thereof.

Formulation and Administration

In certain embodiments, the present disclosure provides a method oftreating a subject (e.g., a human) with a disorder mediated by atherapeutic target (target molecule(s)), e.g., CD3, PSMA, CD19, CXCR5,CD33, PDL1, VEGFR2, cMet, and Ax1, using a composition comprising abispecific antibody compound described herein and a pharmaceuticallyacceptable carrier. In certain embodiments, the amount of bispecificantibody compound described herein in a provided composition is suchthat it is effective as an inhibitor or agonist in a biological sampleor in a subject. In certain embodiments, a provided composition isformulated for administration to a subject in need of such composition.In some embodiments, a provided composition is formulated for parenteralor intravenous administration to a subject.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier, adjuvant, or vehicle that does not destroy the pharmacologicalactivity of the antibody with which it is formulated. Pharmaceuticallyacceptable carriers, adjuvants or vehicles that may be used in thecompositions of this disclosure include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Compositions described herein may be administered orally, parenterally,by inhalation spray, topically, rectally, nasally, buccally, vaginallyor via an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid are used in the preparation ofinjectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including age, body weight, general health, sex, diet, time ofadministration, rate of excretion, drug combination, the judgment of thetreating physician, and the severity of the particular disease beingtreated. The amount of a provided bispecific antibody compound in thecomposition will also depend upon the particular compound in thecomposition.

Uses of Bispecific Antibody Compounds

Bispecific antibody compounds and compositions described herein aregenerally useful for modulating molecules to which the antibodies arespecific. Examples of molecules which may be bound by the bispecificantibody compounds described herein include, but are not limited to,CD3, PSMA, CD19, CXCR5, CD33, PDL1, VEGFR2, cMet, and Ax1, includingcombinations thereof. In one embodiment, the bispecific antibodycompounds described herein bind a pair of antigens selected from thefollowing combinations: CD3-PSMA, CD3-CD19, CD3-CXCR5, CD3-CD33,PDL1-VEGFR2, PDL1-cMet, and PDL1-Ax1.

Thus, in some embodiments, the present disclosure provides a method oftreating disorders associated with detrimental activity of CD3, PSMA,CD19, CXCR5, CD33, PDL1, VEGFR2, cMet, or Ax1, comprising administeringa provided compound or composition.

In one embodiment, the bispecific antibody compounds described hereinbind an antigen or combination of antigens selected from the following:CD3, PSMA, CD19, CXCR5, CD33, PDL1, VEGFR2, cMet, and Ax1, may be usedto treat a subject having a disorder selected from non-Hodgkin lymphoma(NHL), prostate cancer, B-cell lymphoma, acute myeloid leukemia (AML),colon cancer, breast cancer. Modulation of a target molecule(s), e.g.,CD3, PSMA, CD19, CXCR5, CD33, PDL1, VEGFR2, cMet, and/or Ax1, of thebispecific antibody compound described herein means that a change oralternation in the activity of the target molecule(s), e.g., CD3, PSMA,CD19, CXCR5, CD33, PDL1, VEGFR2, cMet, and/or Ax1, has occurred from theadministration of one or more of the bispecific antibody compoundsdescribed herein. Modulation may be an upregulation (increase) or adownregulation (decrease) in the magnitude of the activity or functionof the target molecule(s), e.g., CD3, PSMA, CD19, CXCR5, CD33, PDL1,VEGFR2, cMet, and Ax1. Exemplary activities and functions include e.g.,binding characteristics, enzymatic activity, cell receptor activation,transcriptional activity, and signal transduction.

Diseases and conditions treatable according to the methods using thebispecific antibody compounds described herein include, but are notlimited to, treating or ameliorating cancer or another proliferativedisorder by administration of an effective amount of a bispecificantibody compound described herein to a mammal, e.g., a human in need ofsuch treatment. In some aspects, the disease and conditions to betreated by the methods herein is cancer. Examples of cancers treatedusing the compounds and methods described herein include, but are notlimited to, adrenal cancer, acinic cell carcinoma, acoustic neuroma,acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia,acute erythroid leukemia, acute lymphoblastic leukemia, acutemegakaryoblastic leukemia, acute monocytic leukemia, actue promyelocyticleukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoidodontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm,adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressiveNK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma,alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large celllymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma,angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoidtumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocyticleukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer,bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor,Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma insitu, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma,chondroma, chordoma, choriocarcinoma, choroid plexus papilloma,clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-celllymphoma, cervical cancer, colorectal cancer, Degos disease,desmoplastic small round cell tumor, diffuse large B-cell lymphoma,dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonalcarcinoma, endocrine gland neoplasm, endodermal sinus tumor,enteropathy-associated T-cell lymphoma, esophageal cancer, fetus infetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroidcancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor,gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumorof the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosiscerebri, glucagonoma, gonadoblastoma, granulosa cell tumor,gynandroblastoma, gallbladder cancer, gastric cancer, hairy cellleukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma,hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma,intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna,lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lungcancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma,acute lymphocytic leukemia, acute myelogeous leukemia, chroniclymphocytic leukemia, liver cancer, small cell lung cancer, non-smallcell lung cancer, MALT lymphoma, malignant fibrous histiocytoma,malignant peripheral nerve sheath tumor, malignant triton tumor, mantlecell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia,mediastinal germ cell tumor, medullary carcinoma of the breast,medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkelcell cancer, mesothelioma, metastatic urothelial carcinoma, mixedMullerian tumor, mucinous tumor, multiple myeloma, muscle tissueneoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma,nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma,neuroma, nodular melanoma, ocular cancer, oligoastrocytoma,oligodendroglioma, oncocytoma, optic nerve sheath meningioma, opticnerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor,papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma,pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma,polyembryoma, precursor T-lymphoblastic lymphoma, primary centralnervous system lymphoma, primary effusion lymphoma, preimary peritonealcancer, prostate cancer, pancreatic cancer, pharyngeal cancer,pseudomyxoma periotonei, renal cell carcinoma, renal medullarycarcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter'stransformation, rectal cancer, sarcoma, Schwannomatosis, seminoma,Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cellcarcinoma, skin cancer, small blue round cell tumors, small cellcarcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinaltumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovialsarcoma, Sezary's disease, small intestine cancer, squamous carcinoma,stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroidcancer, transitional cell carcinoma, throat cancer, urachal cancer,urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer,verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginalcancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms'tumor.

In one aspect the diseases and conditions treatable by the according tothe methods using the bispecific antibody compounds described herein areselected from non-Hodgkin lymphoma (NHL), prostate cancer, B-celllymphoma, acite myeloid leukemia (AML), colon cancer, and breast cancer.In one embodiment, the bispecific antibody compounds described hereinare used as bispecific T cell engagers, and are able to exert action onits antigen selectively and direct the human immune system to actagainst a tumor cell.

In one embodiment, a human patient is treated with a bispecific antibodycompounds described herein and a pharmaceutically acceptable carrier,adjuvant, or vehicle, wherein said bispecific antibody compound ispresent in an amount to treat or ameliorate one or more of the diseasesand conditions recited above. In an alternative embodiment, the diseasesand conditions treated or ameliorated by a bispecific antibody compounddescribed herein include, any one of those described above. In oneaspect, the diseases and conditions are selected from non-Hodgkinlymphoma (NHL), prostate cancer, B-cell lymphoma, acite myeloid leukemia(AML), colon cancer, breast cancer, in the patient.

Exemplification

As depicted in the Examples below, in certain exemplary embodiments,bispecific antibody compounds are prepared according to the followinggeneral procedures. It will be appreciated that, although the generalmethods depict the synthesis of certain compounds herein, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all bispecific antibody compounds and subclassesand species of each of these bispecific antibody compounds, as describedherein.

Preparation of Bispecific Antibodies Compounds of Formula I Fc and HingeRegion Removal of Antibodies

Antibodies were buffered exchange into 20 mM Sodium Phosphate (JT Baker3827-01) and 10 mM EDTA (Aldrich E26290) and (1.0 mg) was added to 80 μLPapain Slurry (Thermo Scientific Pierce 20341) with 20 mM Cysteine(Sigma C7352) and incubated in 37° C. for 6.5 h in head to head spinner.Fc fragment and undigested IgG was then removed from the Fab usingprotein A purification via ÅKTA pure chromatography system.

In an alternative, proteolytic digestion of IgG1 allowed generation ofF(ab) proteins. SpeB cysteine protease, FabULOUS (Genovis), was used todigest the hinge region of IgG1 to produce F(ab) and Fc fragments. Adigestion procedure was adopted using 0.1-0.2 U/μg overnight (˜16 h) at37° C. in Dulbecco's phosphate-buffered saline (DPBS) with 1 mMdithiothreitol (DTT). Samples were then either buffer exchanged toremove DTT or diluted to decrease DTT concentration prior to F(ab)purification. Protease was removed by Ni-NTA gravity column, then theflow-through (FT) was subjected to Protein A purification by standardmethods. Protein A FT contain the F(ab) fragment while the Fc and anyundigested IgG1 was retained in the column. For larger scale F(ab)preparations, digestion was performed in a buffer containing 20 mMimidazole, 0.5 M NaCl, and 20 mM sodium phosphate (pH 7.4) with 0.1 mMDTT using 0.1-0.2 U/μg overnight (˜16 h) at 37° C. enabling tandemHisTrap FF (GE) and HiTrap MabSelect SuRe (GE) purification on an ÅKTApure chromatography system. F(ab) purity was assessed by SDS-PAGEanalysis and HIC HPLC. See FIG. 5 and FIG. 6.

SDS-PAGE Analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) wasemployed using NuPAGE Novex 4-12% Bis-Tris Protein Gels with NuPAGE MOPSSDS Running Buffer in a XCell SureLock Mini electrophoresis system. Allsamples (2.5 μg) included NuPAGE LDS Sample Buffer and were heated to95° C. for 5 min prior to loading. Reduced samples also contained NuPAGESample Reducing Agent. Mark12 Unstained Standard (10 μL) was used forestimation of molecular weights. After gel electrophoresis at 125 V for1.5 h, gels were fixed for 5 min and stained with SYPRO Ruby Protein GelStain following the recommended procedures. Imaging was performed with aBio-Rad ChemiDoc MP System and analyzed by Image Lab Software.

SDS-PAGE Analysis Hydrophobic Interaction Chromatography (HIC) HPLC

Analysis by HIC HPLC used a TOSOH TSKgel Butyl-NPR (4.6 mm ID×10 cm, 2.5μm) column at 40° C. on an Agilent 1260 Infinity system. Analytical runswere performed using 50 μg sample with a linear gradient of 0-60% B over30 min: A=50 mM sodium phosphate+1 M ammonium sulfate (pH 7), B=50 mMsodium phosphate+25% isopropanol (pH 7). All data was analyzed usingOpenLAB Software.

General Procedure for Cyclization

Conjugation of FAB¹ and FAB² was enabled through reduction of theinterchain disulfide bonds, followed by reaction with a2,3-dibromomaleimide (DBM) intermediate comprising an azide ordibenzylcyclooctyne (DBCO). Cyclization was then commenced viacopper-free click chemistry. See also FIG. 7 for a generalrepresentation of the approach using αPSMA and αCD3 as an example. The2,3-dibromomaleimide (DBM) intermediates comprising an azide wereprepared in situ by reacting the appropriate DBM-PEG-DBCO linker (e.g.,for αPSMA/αCD3 bispecific antibody compounds described below,DBM-PEG4-DBCO and DBM-PEG8-DBCO were used) with 10-15 equivalents of theappropriate azido-PEG-azide (e.g., for αPSMA/αCD3 bispecific antibodycompounds described below, azido-PEG2-azide was used) for 1 h at roomtemperature (RT). F(ab) proteins (e.g., αPSMA, αCD3) at 5 mg/mL weretypically reduced using 5 or 10 equivalents of DTT for 1 hour at RTfollowed by conjugation with 10 or 15 equivalents DBM linker,respectively, and 7.5% DMSO co-solvent overnight at RT. Excess linkerwas removed by centrifugal filtration. Heavy chain-light chain disulfidebridging was determined to be ˜85% efficient by SDS-PAGE and HIC HPLCanalysis. Cyclization was initiated by mixing the FAB¹-X intermediateand the FAB²-Y intermediate at 5 mg/mL for 24-48 h at either roomtemperature or 37° C. Purity of the antibody and intermediates prior tocyclization was assessed by SDS-PAGE analysis and HIC HPLC. See FIG. 6,FIG. 8, and FIG. 9 for data pertaining to the αCD3 F(ab) and αPSMA F(ab)products. Cyclization products were usually formed with ˜65-95% yielddepending on incubation temperature and time.

Anti-PDL1/Anti-VEGFR2 Bispecific Antibody Compounds

A bispecific antibody compound of Formula I, where FAB¹ is anti-PDL1 andFAB² is anti-VEGFR2 was prepared as follows. The FAB¹ (anti-PDL1antibody) comprised variable regions having amino acid sequencescorresponding to SEQ ID Nos: 1 and 2. The FAB² (anti-VEGFR2 antibody)comprised variable regions having amino acid sequences corresponding toSEQ ID Nos: 3 and 4.

Following Fc removal of anti-PDL1 and anti-VEGFR2, each antibody (1-10mg) was added to separate 15 mL filter centrifuge tubes (Millipore,UFC903024) and an appropriate volume of a 50 mM sodium phosphate,150 mMNaCl, 5 mM EDTA, pH 7.7 buffer was added to the 50 mL mark on the tube.The tubes were centrifuged at 3,000 RPM for 20 min at 22° C. Theantibodies were then transferred into separate 1.5 mL plastic vials andconcentrations were confirmed using Nanodrop (Fisher, ND-2000 UV-VisSpectrophotometer). The final antibody concentrations were up to 5mg/mL. In this example, anti-PDL1 was used as FAB¹ and anti-VEGFR2 wasused as FAB².

A stock solution of 1 mg/mL TCEP ((tris(2-carboxyethyl)phosphine)),Sigma-Aldrich, C4706) in pH 8.0 PBS (1 mM EDTA) buffer was prepared.Five equivalents of TCEP was added to FAB¹ and the mixture was shakenand incubated at room temperature for 1 h. TCEP was separated from thereduced FAB¹ using a NAP-5 (GE17-0853-02) desalting column.

A stock solution of Dibromo-DBCO (2,3-dibromomaleic anhydride; ClickChemistry Tools, A108-100) in DMSO (Sigma-Aldrich, 472301) was preparedand 1 equivalent of Dibromo-DBCO in DMSO was added to the FAB¹ sample.The final volume of DMSO in the sample was about 5-9% (v/v). Theconjugation reaction between Dibromo-DBCO and FAB¹ was conducted for 1 hat RT under mixing by carousel. This step was repeated two more times.The final concentration for the Dibromo-DBCO was 3 equivalents of FAB¹.

The Dibromo-azide (1 equivalents) in DMSO was added to the FAB¹ sample.Final volume of DMSO in antibody sample is about 5-9% (v/v). Theconjugation reaction was conducted for 1 h at RT under mixing bycarousel, this step was repeated for two more times with finalconcentration for the Dibromo-DBCO being 3 equivalents.

Each sample was placed into a separate 15 mL filter centrifuge tube(Millipore, UFC903024) and added an appropriate volume of 1×DPBS plus10% DMSO (Corning, 21-031-CM, no calcium or magnesium) buffer to the 50mL mark on the tube. The samples were centrifuged at 3,000 RPM for 20min at 22° C. The wash step was repeated once more. Then an appropriatevolume of 1×DPBS (Corning, 21-031-CM, no calcium or magnesium) bufferwas added to the 50 mL mark on the tube. The samples were centrifuged at3,000 RPM for 20 min at 22° C. After wash, the samples was transferredinto separate 1.5 mL plastic vials and placed in refrigerator (5° C.) orwas used for click step.

For each sample to be analyzed, 20 μL at a concentration of 0.6 mg/mL isrequired. Follow the established protocols for running SDS-PAGE gels(RTP AD001-01 and AD002-01) See FIG. 2, where 1) represents FAB¹-DBCO;2) represents FAB²-azide; and 3) represents the bispecific antibodycompound of Formula I (Click product from FAB¹-DBCO and FAB²-azide.

Specific examples are provided below.

1-(2-(2-azidoethoxy)ethyl)-3,4-dibromo-1H-pyrrole-2,5-dione

To 2.5 g of 3,4-dibromo-1H-pyrrole-2,5-dione (10 mmol) and 1 g of NMM in60 mL of THF, MeOCOCl (10 mmol, 940 mg in 10 ml DCM) was added dropwise,stirred for 20 min, then the reaction solution was diluted with 60 mL ofDCM, washed 3 time by water, the organic phase was stirred by sodiumsulfate anhydrous, concentrated, 2.65 g of methyl3,4-dibromo-2,5-dioxo-2H-pyrrole-1(5H)-carboxylate was obtained. To 311mg, 1 mmol of this compound, 2-(2-azidoethoxy)ethanamine (130 mg, 1mmol) and 5 mL DCM was added, TLC shown the reaction finished in 20 min,then extracted by DCM and brine, washed by NH₄Cl solution, dried onsodium sulfate anhydrous, and then concentrated for column purification,flashed by 2:1 hexane and ethyl ethylate, 230 mg of1-(2-(2-azidoethoxy)ethyl)-3,4-dibromo-1H-pyrrole-2,5-dione obtained.¹HNMR: 3.32 ppm (t, J=5.0 Hz, 1H), 3.40 ppm (t, J=5.0 Hz, 1H), 3.50 ppm(q, J=5.0 Hz, 1H), 3.62 ppm (t, J=5.0 Hz, 1H), 3.63-3.69 ppm (m, 3H),3.84 ppm (t, J=5 hz, 1H). Fw: 365.9, C₈H₈Br₂N₄O₃; Mass Peaks (1:2:1):366.9, 368.9, 370.9.

N-(3-(1-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-1,9-dihydro-8H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)-1-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amide(102)

To a solution of dibromo-maleimide-PEG2-dibenzocyclooctyne (1.0 mg, 1.0equivalent, 100) in DMSO (0.13 mL) was added azido-PEG2-azide (1.4 mg,5.0 equivalent, 101) in DMSO (0.7 mL). The mixture was stirred at roomfor 1 hr. The reaction was completed as indicated by LC/MS. Molecularweight of the resulting dibromo-maleimide-azide 102 was 961.1 g/mol.

Dibenzocyclooctane-PEG4-maleimide-FAB¹

A stock solution of Dibromo-DBCO (2,3-dibromomaleic anhydride; ClickChemistry Tools, A108-100) in DMSO (Sigma-Aldrich, 472301) was preparedand 1 equivalent of Dibromo-DBCO in DMSO was added to the FAB¹ sample.The final volume of DMSO in the antibody sample was about 5-9% (v/v).The conjugation reaction was conducted for 1 hour at room temperatureunder mixing by carousel. This step was repeated two more times. Thefinal concentration for the Dibromo-DBCO was 3 equivalent of FAB¹.

The sample was placed into a separate 15 mL filter centrifuge tube(Millipore, UFC903024) and added an appropriate volume of 1×DPBS plus10% DMSO (Corning, 21-031-CM, no calcium or magnesium) buffer was addedto the 50 mL mark on the tube. The sample was centrifuged at 3,000 RPMfor 20 minutes at 22° C. The wash step was repeated once more. Then anappropriate volume of 1×DPBS (Corning, 21-031-CM, no calcium ormagnesium) buffer was added to the 50 mL mark on the tube. The samplewere centrifuged at 3,000 RPM for 20 minutes at 22° C. After wash, thesample was transferred into separate 1.5 mL plastic vials and placed inrefrigerator (5° C.) to afford 103.

Azido-PEG2-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)-PEG4-maleimide-FAB²

The Dibromo-azide 102 (1 equivalents) in DMSO was added to the FAB²sample. The final volume of DMSO in the antibody sample was about 5-9%(v/v). The conjugation reaction was conducted for 1 hour at roomtemperature under mixing by carousel. This step was repeated two moretimes. The final concentration for the Dibromo-DBCO was 3 equivalent ofFAB².

The sample was placed into a separate 15 mL filter centrifuge tube(Millipore, UFC903024) and an appropriate volume of 1×DPBS plus 10% DMSO(Corning, 21-031-CM, no calcium or magnesium) buffer was added to the 50mL mark on the tube. The sample was centrifuged at 3,000 RPM for 20minutes at 22° C. The wash step was repeated once more. Then anappropriate volume of 1×DPBS (Corning, 21-031-CM, no calcium ormagnesium) buffer was added to the 50 mL mark on the tube. The samplewas centrifuged at 3,000 RPM for 20 minutes at 22° C. After wash, thesample was transferred into separate 1.5 mL plastic vials and placed inrefrigerator (5° C.) to afford 104.

PEG4 Anti-PDL1/PEG 4Anti-VEGFR2 Bispecific Antibody Compound

To 103 (500 μg) in PBS (5.0 mg/mL) was added 104 fragment (500 μg) inPBS (5.0 mg/mL). The reaction was conducted for overnight at roomtemperature under mixing by carousel. The mixture was subjected to SECanalysis.

Agilent 1200 HPLC using a Tskgel Buytl-NPR 4.6 mm×10 cm 2.5 um was usedto analyze the FAB¹-DBCO, FAB²-azide and bispecific antibody compoundproduct of Formula I. Buffer A: 50 mM NaH₂PO₄, 1.5M (NH₄)₂ SO₄ pH 7.0and Buffer B: 50 mM NaH₂PO₄ pH 7.0+25% IPA.

See FIG. 3. MS found molecular weight of 95954.00, theoretical 95955.00for 105.

Bispecific antibody compounds of Formula I were purified viaSize-exclusion chromatography (SEC) using an Agilent 1200 HPLC using aTSK gel SuperSW3000 column (4.6 mm ID×30 cm, 4 μm). Buffer was 0.2 Mpotassium phosphate, 0.25 M KCl, pH 6.2.

αPSMA/αCD3 Bispecific Antibody Compounds General Procedure forαPSMA-PEG4/αCD3-PEG4 (106), αPSMA-PEG4/αCD3-PEG8 (107),αPSMA-PEG8/αCD3-PEG4 (108) and αPSMA-PEG8/αCD3-PEG8 (109)

αPSMA-PEG4/αCD3-PEG4 (106), αPSMA-PEG4/αCD3-PEG8 (107),αPSMA-PEG8/αCD3-PEG4 (108) and αPSMA-PEG8/αCD3-PEG8 (109) weresynthesized according to the methods described above, and using theappropriate starting materials. See also FIG. 7 for a generalrepresentation of the approach. For example, the azide linker wasprepared in situ by reacting DBM-PEG4-DBCO and DBM-PEG8-DBCO linker with10-15 equivalents azido-PEG2-azide for 1 h at room temperature (RT).F(ab) proteins (5 mg/mL) were typically reduced using 5 or 10equivalents of DTT for 1 h at RT followed by conjugation with 10 or 15equivalents DBM linker, respectively, and 7.5% DMSO co-solvent overnightat RT. Excess linker was removed by centrifugal filtration. Heavychain-light chain disulfide bridging was determined to be ˜85% efficientby SDS-PAGE and HIC HPLC analysis. Cyclization was initiated by mixingthe αPSMA F(ab) intermediate and the αCD3 F(ab) intermediate at 5 mg/mLfor 24-48 h at either room temperature or 37° C. Purity of the antibodyand intermediates prior to cyclization were assessed by SDS-PAGEanalysis and HIC HPLC. See FIG. 6, FIG. 8, and FIG. 9. The yield of thefinal products were ˜65-95% yield depending on incubation temperatureand time. Purity of bispecific products 106, 107, 108, and 109 wereassessed by SDS-PAGE and shown in FIG. 10.

In Vitro Affinity Measurements Using Octet Red

Sensors AR2G were used to measure antigen interactions with 105 on theOctet Red (ForteBio, Inc.) In short, the measurement scheme was asfollows: 300 seconds baseline; 300 seconds loading of 10 μg/ml AntigenA, 120 seconds baseline; 300 seconds 105; 300 seconds dissociation; 300seconds Antigen B and 300 seconds dissociation (FIG. 4). Sensorhydration and baseline- and dissociation measurements were performed inPBS. As described in FIG. 4, each Fab fragment was able to maintainantigen binding.

Luminescence Cytotoxicity Assay with Bispecific Antibody Compounds 108and 109

Firefly luciferase transduced prostate cancer target cell lines wereused for cytotoxicity assays, LNCaP (ATCC® CRL-1740™), PSMA+(cultured inRPMI-1640+10% non-heat-inactivated FBS+0.5 μg/mL Puromycin) and PC-3(ATCC® CRL-1435™), PSMA-(cultured in RPMI-1640+10% heat-inactivatedFBS+1.0 μg/mL Puromycin). Cells were harvested with TrypLE (ThermoFisherScientific) then resuspended in fresh RPMI-1640+10% heat-inactivated FBSand plated at 4,000 cells/well in 100 μL. After overnight incubation at37° C. in a humidified 5% CO₂ incubator, serial dilutions of FAB andbispecific antibody compounds of Formula I in RPMI-1640+10%heat-inactivated medium (50 μL) were added to the assay plates at theindicated concentrations. Freshly thawed peripheral blood mononuclearcells (PBMCs) were washed with media and added to the assay plates at40,000 cells in 50 μL to obtain an effector:target ratio of 10:1. After4 days incubation, 90 μL was removed from assay plates and 90 μL ONE GloLuciferase Assay Reagent (Promega #E6120) was mixed with the samples andincubated at room temperature for 10 min. Samples were transferred towhite 96-well flat bottom plates for luminescence measurements using aPerkinElmer EnSpire multimode plate reader. Data was analyzed usingGraphPad Prism software. Cell killing was observed with compounds 108and 109 at PSMA+ LNCaP cells with a potency of −1 nM. Parental F(ab)proteins did not show cytotoxic activity. No bispecific antibodycompound of Formula I-mediated cell killing was observed for PSMA-PC-3cells. The data indicate that PSMA-directed cytotoxicity with bispecificantibody compound of Formula I was achieved using PBMCs ateffector:target 10:1.

1.-16. (canceled)
 17. A bispecific antibody compound having the FormulaII, IIa, III, or IIIa:

wherein: FAB¹ represents a first Fab fragment; FAB² represents a secondFab fragment; R¹ and R² are each independently a substituted alkyl; ringA is a substituted carbocyclyl or substituted heterocyclyl; R^(a) andR^(b) are each independently selected from

Q, T, and V are each independently N or CH; “

” indicates the points of attachment to FAB¹ or FAB²; and “------”indicates the point of attachment to R¹ or R².
 18. The bispecificantibody compound of claim 1, wherein ring A is a substituted bicyclicor polycyclic carbocyclyl or a substituted polycyclic heterocyclyl. 19.The bispecific antibody compound of claim 1, wherein: ring A is

the dashed bonds indicate the points of attachment to the triazolyl; andthe wavy bond indicates the attachment to R².
 20. The bispecificantibody compound of claim 1, wherein R¹ and R² are each independently a(C₂-C₃₀)alkyl or a substituted (C₂-C₃₀)alkyl.
 21. The bispecificantibody compound of claim 4, wherein R¹ and R² are each independently a(C₂-C₃₀)alkyl or a substituted (C₂-C₃₀)alkyl interrupted with one ormore heteroatoms selected from N, O, and S.
 22. The bispecific antibodycompound of claim 1, wherein R¹ and R² are each independently a(C₂-C₃₀)alkyl or a substituted (C₂-C₃₀)alkyl.
 23. The bispecificantibody compound of claim 6, wherein R¹ and R² are each independently a(C₂-C₃₀)alkyl or a substituted (C₂-C₃₀)alkyl interrupted with one ormore heteroatoms selected from N and O.
 24. The bispecific antibodycompound of claim 1, wherein R¹ and R² are each independently a(C₂-C₃₀)alkyl interrupted with at least one O and at least one N, andsubstituted with at least one oxo.
 25. The bispecific antibody compoundof claim 1, wherein R¹ and R² are each independently selected from

wherein, the wavy lines indicate the points of attachment to R^(a) orR^(b); the dashed lines indicated the points of attachment to thetriazolyl or ring A; and p and w independently are integers from 1 to 8.26. The bispecific antibody compound of claim 1, wherein R¹ is selectedfrom

wherein, the wavy lines indicate the points of attachment to R^(a); andthe dashed lines indicated the points of attachment to the triazolyl.27. The bispecific antibody compound of claim 1, wherein R² is selectedfrom

wherein, the wavy lines indicate the points of attachment to R^(a); andthe dashed lines indicated the points of attachment to the triazolyl orring A.
 28. The bispecific antibody compound of claim 1, wherein R^(a)and R^(b) are bound to FAB¹ and FAB² through native cysteines of FAB¹and FAB².
 29. The bispecific antibody compound of claim 1, wherein FAB¹and FAB² do not comprise a hinge region.
 30. The bispecific antibodycompound of claim 1, wherein the bispecific antibody compound binds to atarget molecule selected from the group consisting of CD3, PSMA, DC19,CXCR5, CD33, PDL1, VEGFR2, cMet, and Ax1.
 31. A pharmaceuticalcomposition comprising the bispecific antibody compound of claim 1 and apharmaceutically acceptable carrier.
 32. A method of treating a diseaseor condition effected by the modulation of CD3, PSMA, CD19, CXCR5, CD33,PDL1, VEGFR2, cMet, or Ax1, comprising administering to a subject inneed thereof, a bispecific antibody compound of claim 1, or apharmaceutically acceptable salt thereof.